Regulation of the lateral diffusion of WGA-labeled glycoconjugates in human leukocytes

Abstract

The regulation of the membrane mobility of glycoconjugates in human polymorphonuclear leukocytes (PMNL) was studied by comparing adult PMNL with promyelocytic HL60 cells before and after stimulation of differentiation in HL60 cells with phorbol-myristate acetate (PMA) with respect to lateral diffusion of wheat germ agglutinin (WGA)-labeled glycoconjugates. For this purpose we developed a novel variant of microscope equipment for the study of fluorescence recovery after photobleaching (FRAP) and continuous fluorescence microphotolysis (CFM) using a mini-computer for handling of shutters, data acquisition, and calculations. This equipment is presented in the report. We found that PMA-induced differentiation in HL60 cells reduced the lateral diffusion coefficient (D) of WGA-labeled membrane entities from about 1.5 to 1.0×10⁻¹⁰ cm²/s, which was close to that found for adult blood PMNL, i.e., 1–1.2×10⁻¹⁰ cm²/s. The lateral mobility (D×10¹⁰) of succinylated WGA (S-WGA) was 2.3 and 1.7 cm²/s in undifferentiated and PMA-differentiated HL60 cells, respectively, indicating that WGA might have cross-linked membrane receptors, resulting in the slower diffusion. The results are discussed in relation to the effect of phagocyte maturation on the mobility of membrane components.